Chromatin Structure In Regulation Of Mammalian Gene Expression

NLI/LDB1是一种广泛表达的核因子，是增强子与靶基因长程相互作用的潜在介导物。我们已经证明了LDB1和红系伙伴SC1、GATA-1和LMO2形成了β-珠蛋白转录和基因与β-珠蛋白LCR增强子之间的染色质环所必需的复合体。在最近的工作中，我们发现具有伸长活性的Ser-2P polII及其激酶P-TEFb共同占据LCR和β-珠蛋白启动子，其动力学与LDB1相同，并且LDB1是它们招募所必需的。使用纯合的小鼠缺失LCR，我们证明这些成分被招募到β-珠蛋白启动子，而不是LCR。然而，LDB1复合体的占有率不足以进行高水平的转录，这突显了LCR/基因远程相互作用的重要性。此外，使用RNAi降低LDB1水平会导致该基因座无法从核外围迁移出去，这是在核转录工厂实现β-珠蛋白的强劲转录所必需的。这些数据表明，染色质环的形成促进了高水平的转录，因为它是介导核内迁移所必需的。 在其他实验中，我们探索了绝缘体阻断增强子的机制。我们发现，人β-珠蛋白HS5是依赖CTCF的鸡HS4绝缘体的同源物，具有内在的、可移植的增强子阻断活性，这通过染色质环的形成而明显。事实上，在围绕β-珠蛋白LCR的两个CTCF位点之间的循环在拓扑上将LCR与其靶标珠蛋白基因分离，并使其增强子功能失效。为了研究CTCF位点的环化活性是否是基因组中这些位点的一般特性，我们在11号染色体上超过2Mb的CTCF/粘附素位点上进行了染色质构象捕获(3C)，包括β-珠蛋白基因座和两侧的嗅觉受体基因。我们发现，这些位点之间的相互作用频率是高度细胞类型特异性的，这表明在缺乏珠蛋白基因活性的情况下，CTCF位点的组织更加密集。为了评估CTCF依赖环的功能，我们在主动转录胎儿珠蛋白基因的细胞中使用RNAi减少了CTCF。随着受抑的组蛋白标记的获得，γ-珠蛋白的表达显著减少。这些数据为人类基因组中CTCF位点的调控重要性提供了一个例子，以定义功能域并通过环的形成证明CTCF/粘附素位点的一般染色体组织作用。

NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long range interaction between enhancers and target genes. We have shown that Ldb1 and erythroid partners SCL, GATA-1 and LMO2 form a complex that is required for beta-globin transcription and for chromatin looping between the gene and the beta-globin LCR enhancer. In recent work, we have discovered that elongation competent Ser-2P pol II and its kinase P-TEFb co-occupy the LCR and beta-globin promoter with the same kinetics as Ldb1 and that Ldb1 is required for their recruitment. Using mice homozygous for a deletion of the LCR, we demonstrated that these components are recruited to the beta-globin promoter independent of the LCR. However, occupancy by the Ldb1 complex is insufficient for high level transcription, highlighting the importance of the LCR/gene long range interaction. In addition, reduction of Ldb1 levels using RNAi results in the inability of the locus to migrate away from the nuclear periphery which is necessary to achieve robust transcription of beta-globin in nuclear transcription factories. These data suggest chromatin loop formation promotes high level transcription because it is required for mediating intra-nuclear migration. In other experiments, we explored the mechanisms underlying enhancer blocking by insulators. We found that human beta-globin HS5, the orthologue of the CTCF dependent chicken HS4 insulator, has intrinsic, portable enhancer blocking activity that is manifest though chromatin loop formation. In fact, looping between two CTCF sites engineered to surround the beta-globin LCR topologically isolates the LCR from its target globin genes and nullifies its enhancer function. To investigate whether the looping activity of CTCF sites is a general property of these sites in the genome, we carried out chromatin conformation capture (3C) on CTCF/cohesin sites over 2 Mb on chromosome 11 encompassing the beta-globin locus and flanking olfactory receptor genes. We found that the interaction frequencies among the sites are highly cell type specific revealing a more densely clustered organization of CTCF sites in the absence of globin gene activity. To assess the function of the CTCF dependent loops, we reduced CTCF using RNAi in cells actively transcribing the fetal -globin genes. Expression of gamma-globin was strongly reduced in concert with the acquisition of repressive histone marks in the locus. These data provide an example of the regulatory importance of CTCF sites in the human genome to define functional domains and document a general chromosome organizational role for CTCF/cohesin sites through loop formation.